Dynamics of proximal signaling events after
TCR/CD8-mediated induction of proliferation or apoptosis in
mature T-cells
Dissertation
zur Erlangung des akademischen Grades
doctor rerum naturalium (Dr. rer. nat.)
genehmigt durch die Fakultät für Naturwissenschaften der Otto-von-Guericke-Universität Magdeburg
von Dipl.-Biol. Xiaoqian Wang
geb. am 24.12.1973 in Shaanxi, China
Gutachter: Prof. Dr. Burkhart Schraven Prof. Dr. Ottmar Janssen
eingereicht am: 1. July 2009 verteidigt am: 30. September 2009
Table of content
1. Introduction 6 1.1. T cell development 6 1.2. T cell activation 8 1.2.1. The TCR 8 1.2.2. TCR signaling 9 1.2.2.1. Src kinases 9 1.2.2.2. ZAP-70 10 1.2.2.3. Adaptor proteins 111.2.2.4. PLCγ1 activation and calcium flux 13
1.2.2.5. Ras-Raf-ERK1/2 activation 14
1.2.3. The role of TCR signaling in thymocyte development 15 1.2.4. Negative regulation of antigen receptor signaling in T cells 17 1.2.4.1. PTPases (Protein Tyrosine Phosphatases) 17 1.2.4.2. Negative regulation of T cell activation by ubiquitination and
degradation 19
1.3. T cell apoptosis 20
1.3.1. Two signaling pathways leading to apoptosis 21
1.4 Aims of the present research 23
2. Materials and methods 24
2.1. Materials 24 2.1.1. Instruments 24 2.1.2. Plastic ware 25 2.1.3. Kits 25 2.1.4. Chemical Reagents 25 2.1.5. Antibodies 27
2.1.5.1. Antibodies for stimulation 27
2.1.5.2. Antibodies for flow cytometric analysis 27 2.1.5.3. Antibodies for Western blotting and Immunoprecipitation
(IP) 27
2.1.5.5. Secondary antibodies 29
2.1.6. Streptamers 29
2.1.7. Mouse strains 29
2.2. Methods 29
2.2.1. Animal experimentation 29
2.2.1.1. Mouse condition and handling 29 2.2.1.2. Genomic DNA isolation from mouse tail 29
2.2.1.3. Genotyping of OT1-transgenic mice strain by PCR
(Polymerase-Chain Reaction) 30
2.2.1.4. Gel electrophoresis of nucleic acids 31
2.2.2. T cell purification 31
2.2.2.1. Preparation of single cell suspension from mice spleens 31
2.2.2.2. Cell counting 32
2.2.2.3. Purification of CD8+ T cell 32
2.2.3. T cell stimulation in vitro 32 2.2.3.1. T cell stimulation in vitro with OT1-streptamers 32
2.2.3.2. CD3/CD8 mAbs stimulation 33
2.2.3.3. Plate-bound CD3/CD8 mAbs stimulation 33 2.2.4. Surface staining and FACS (Fluorescence Activated Cell Sorting)
analysis 33
2.2.4.1. Extracellular staining 33
2.2.4.2. TCR internalization assay 34
2.2.5. Proliferation assay 34
2.2.6. Apoptosis assay 34
2.2.7. Caspase-3 activity assay 34
2.2.8. Calcium flux determination 35
2.2.9. Immunoblotting 35
2.2.9.1. Cell lysis 35
2.2.9.2. Protein concentration measurement 36 2.2.9.3. SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel
Electrophoresis) 36 2.2.9.4. Western blotting analysis and immunoblotting 37
2.2.9.5. Immunoprecipitation 38
3. Results 40 3.1. TCR triggering with soluble CD3/8 mAbs causes apoptosis while
OT1-streptamers induce proliferation 41
3.1.1. Quality control of OT1-streptamers 41 3.1.2. TCR triggering with OT1-streptamers leads to T cell proliferation 43 3.1.3. The susceptibility of CD8+ T cells to apoptosis in response to
antibody stimulation 45
3.1.3.1. Antibody stimulation induces apoptosis 45 3.1.3.2. The intrinsic versus extrinsic signaling pathways of apoptosis
and their involvement in TCR induced apoptosis 49 3.1.4. OT1-streptamer stimulation induces CD8+ T cell activation and
differentiation into effector T cells 53
3.2. Analysis of signaling pathways regulating proliferation after TCR
stimulation 56 3.2.1. Streptamers induce sustained activation of ERK1/2 56
3.2.2. Activation-induced degradation of ζ chain, Lck, and ZAP-70 after antibody stimulation contributes to transient ERK1/2
activation 58
3.2.3. Kinetics of LAT phosphorylation 65
3.2.4. OT1-streptamers induce a weak calcium mobilization but
sustained phophorylation of PLCγ1 66
3.3. Spatial compartmentalization and distinct subcellular localization of
ERK1/2 69 3.4. The conversion from apoptosis to cell survival by addition of
lysosome inhibitor in response to antibody stimulation 78
4. Discussion 83
4.1. Soluble CD3/CD8 mAbs induce apoptosis via Bim-caspase-
mediated pathway 83
4.2. Molecular mechanism leading to sustained ERK1/2 activation 86 4.3. The divergence of signals for T cell activation at the level of LAT /
PLCγ1 88 4.4. Negative regulation of T cell activation 90
5. References 98
6. Abbreviations 109
7. Acknowledgements 110
8. Summary 111
1.
Introduction
The function of the immune system is to prevent and eradicate infections by means of layered defenses of increasing specificity. Physical barriers prevent pathogens from entering the organism at the first stage. However, if a pathogen breaches these barriers, the innate immune system provides an immediate, but non-specific response, called innate immune response. Only when the innate immune responses are bypassed an adaptive immune response is required. The adaptive immune response is long-lived and specialized protective imunity and is triggered when microbes and microbial antigens enter into secondary lymphoid organs following a simple diffusion or capture by APCs (Antigen Presenting Cells). Secondary lymphoid organs, including lymph nodes, spleen and mucosal-associated lymphoid tissues, form a complex system that supports the interaction between antigen and antigen-specific lymphocytes. There are two types of adaptive immunity, humoral immunity and cell mediated immunity. Humoral immunity is mediated by antibodies produced by B lymphocytes. Antibodies are secreted into the circulation and mucosal fluids, where they participate in host defense to eliminate microbes in different ways including neutralization, opsonization, and complement activation. Cell mediated immunity refers to the host defense against intracellular microbes. It involves the activation of macrophages, NK (Natural Killer) cells, T cells which subsequently differentiated into antigen-specific CTLs (Cytotoxic T Lymphocytes), and the release of various cytokines in response to an antigen [1]. 1.1. T cell development
T cells arise from hematopoietic stem cells and the progenitors migrate from the bone marrow to the thymus, where they develop into naive T cells. Upon entering into the thymus, the progenitors lack the expression of the co-receptors CD4 and CD8, and therefore, are called DN (Double Negative) thymocytes. The development of thymocytes follows sequential steps that are defined by the expression of CD4 and CD8 and the developing thymocytes are divided into four major subsets, namely, CD4 -CD8- (DN), CD4+CD8+ (DP-Double Positive), CD4-CD8+ (SP-Single Positive) and CD4+CD8- (SP) thymocytes. Based on the cell-surface expression of CD25 and CD44, the DN population can be further divided into the DN1, DN2, DN3 and DN4 stages (Figure 1) [2]. It has also been demonstrated that the different stages of thymocyte development occur within defined thymic regions. The hematopoietic progenitors enter the thymus through post-capillary venules and mature to CD44+ (DN1), cells. As they
migrate outward towards the thymic capsule in response to chemokine signaling, they upregulate CD25 (DN2) and then downregulate CD44 (DN3) [3]. At this stage, the TCR (T Cell Receptor) β chain is expressed and assembles with the invariant pre-Tα (pre-TCR α) chain and the CD3 signal transducing molecules. This complex is called the pre-TCR. The failure of the formation of a pre-TCR complex leads to cell death by apoptosis. Pre-TCR signaling leads to the inhibition of further TCR β chain rearrangement (allelic exclusion), cell proliferation and the expression of CD4 and CD8. This process is called β-selection (Figure 1). The rearrangement of the TCR α chain occurs at the DP stage and results in the expression of a mature and functional TCR on the cell surface. DP cells undergo positive or negative selection. Positive selection occurs in the cortex and is triggered by weak interactions between the mature TCR and MHC–self peptide complexes expressed on cTECs (cortical Thymic Epithelial Cells). In response to CCR7 signaling, positively selected thymocytes migrate to the medulla, which is the main site for negative selection [4]. Negative selection involves strong interactions between the TCR and MHC-self peptides displayed on medulla epithelial cells and DCs (Dendritic Cells) within the thymus, which results in apoptosis. Negative selection protects the body from possible autoimmunity by removing T cells that recognize self peptides with high affinity (Figure 1).
Figure 1. Thymic T cell development
Hematopoeitic progenitors enter the thymus and differentiate into DN cells. Thymocyte maturation and differentiation is characterized by the expression of different cell-surface markers, including CD4, CD8, CD44 and CD25 and the TCR. Thymocytes undergo positive selection in the cortex through the interaction between the TCR and MHC–self peptide complexes expressed on cTECs (cortical Thymic Epithelial Cells). The positively selected thymocytes are then screened for reactivity to tissue restricted self peptides by means of negative selection.
1.2. T cell activation
Naive T cells constantly re-circulate throughout the peripheral lymphoid organs to scan for foreign antigens expressed on APCs, such as B cells, dendritic cells, and macrophages. Specific recognition of antigen by the TCR initiates an adaptive immune response [5, 6].
1.2.1. The TCR
The majority of T cells bear TCRs composed of an α chain and a β chain, which are transmembrane proteins. The extracellular portion of each chain consists of two domains, resembling immunoglobulin V and C domains, respectively. The juxtaposition of the immunoglobulin V domains in both α chain and β chain forms the antigen binding site. In addition to the α and β chains, the αβTCR contains the signal-transducing CD3 complex and the homodimer ζ chains [7]. The CD3 complex consists of one CD3γ, one CD3δ, and two CD3ε subunits (Figure 2). The association between the αβ heterodimers, the CD3 complex and the ζ chains is important for T cell activation because the intracellular domains of the α- and the β- chains are short and do not contain signaling motifs. Hence, after the interaction with antigen/MHC, the CD3 complex and ζ chains transmit the signal into the cytoplasm of the T cell to trigger T cell activation.
Signaling from the TCR complex depends on the presence of ITAMs (Immuno receptor Tyrosine-based Activation Motifs), which are present in the cytoplasmic tails of both the CD3 complex and ζ chains. ITAMs are composed of two tyrosine residues which are separated by 6-8 amino acids. Although each of the CD3 subunits contains at least one ITAM, the ζ chains are believed to be the dominant component during T cell signaling because they contain 6 of the 10 ITAMs (three in each ζ chain) [8].
Figure 2. The TCR complex
The TCR complex is composed of the highly variable, antigen-binding TCR heterodimer (α and β chain) and the invariant signaling proteins CD3γ, CD3δ, CD3ε and ζ chain, which are responsible for signal transduction. ITAMs (Immuno receptor Tyrosine-based Activation Motifs) are indicated by white boxes.
Following TCR engagement, one of the first detectable biochemical events is the phosphorylation of the ITAMs. The multiple ITAMs in the ζ chains confer flexibility in signaling [9]. By using sequence-specific phosphotyrosine antibodies, Kersh et al. found that the ζ chains undergo a series of ordered phosphorylation events upon TCR engagement [10, 11]. Complete phosphorylation depends on the nature of the TCR ligand. For example, recognition of a less potent ligand only leads to phosphorylation of a subset of tyrosines on the ζ chains, and complete phosphorylation of the ITAMs is only observed with a strong agonist ligand that fully activates the T cell.
1.2.2. TCR signaling
It is known that tyrosine phosphorylation in ITAMs is the first event after TCR ligation and that it is mediated by Src-family tyrosine kinases (Lck, Fyn). Phosphorylation of the ITAMs allows their interaction with the SH2 domains of ZAP-70 (Zeta-chain-Associated Protein kinase 70) which is important for transmitting the signal onwards (see below). Once bound to ITAMs, ZAP-70 is activated and further contributes to the phosphorylation of multiple downstream molecules, such as LAT (Linker for Activation of T cells), SLP-76 (SH2 domain containing Leukocyte Protein of 76 kDa) and PLCγ1 (Phospholipase C γ1). This signaling cascade initiates calcium flux and MAP kinase activation and ultimately culminates in proliferation and differentiation of T cells.
1.2.2.1. Src kinases
In contrast to the classical receptor tyrosine kinases, the TCR complex does not possess any intrinsic enzymatic activity. However, ITAMs in the TCR complex can be phosphorylated by Src kinases. The phosphorylated ITAMs further transduce the activation signals from the membrane to the nucleus. Therefore, ITAM phosphorylation via Src kinases is the first crucial biological event following TCR engagement. T cells primarily express the Src kinases Lck and Fyn, both of which contain N-terminal lipid modifications (targeting them to the membrane), a unique region, an SH3 domain, an SH2 domain, a proline rich region, and a tyrosine kinase domain. Lck constitutively interacts with either the CD4 or the CD8 coreceptor via a dicysteine motif present in its unique domain [12]. The enzymatic activity of the Src kinase is regulated by the phosphorylation of two tyrosines. Tyrosine 394 (Y394) is located within the kinase domain and is phosphorylated when Lck is active
(autophosphorylation). Tyrosine 505 (Y505) lies in the C-terminal region and has a negative regulatory function. In resting T cells, Y505 is phosphorylated and interacts with the SH2 domain of Lck, thereby leading to a closed and inactive conformation of this kinase [13, 14]. Phosphorylation of Y505 is regulated in two steps. The constitutive phosphorylation of Y505 in resting T cells is mediated by the protein tyrosine kinase Csk (COOH-terminal Src Kinase). TCR engagement triggers the dissociation of Csk from the plasma membrane and the dephosphorylation of Y505 by the protein tyrosine phosphatase CD45, leading to the formation of an open conformation of Lck and its activation (Figure 3). Therefore, Csk is considered to be a critical negative regulator of Lck while CD45 is regarded as a positive one following TCR engagement [15].
1.2.2.2. ZAP-70
Once the ITAMs within the TCR complex are phosphorylated by Src-family kinases, they will recruit the tyrosine kinase ZAP-70 to the plasma membrane. ZAP-70 is a 70 kDa protein expressed throughout thymocyte development and at high level in peripheral T cells. ZAP-70 deficiency leads to a block in T cell development at the transition from the DP to the SP stage [16, 17]. The SH2 domains in ZAP-70 binds to the phosporylated tyrosines in the ITAMs of the TCR complex and thus targets
ZAP-Figure 3. Regulation of Lck activity by CD45 and Csk
Lck activation is regulated by both positive and negative mechanisms. In resting T cells, Lck is deactivated through Csk mediated phosphorylation of its negative regulatory tyrosine Y505. TCR
stimulation induces the dissociation of Csk from its substrate and Lck is in a primed state. CD45 can dephosphorylate both autocatalytic and inhibitory tyrosines of Lck while the primed Lck is able to undergo autophosphorylation of Y394 generating an active kinase that subsequently
70 to the plasma membrane [18]. Once ZAP-70 is targeted to the plasma membrane, it undergoes tyrosine phosphorylation either by autophosphorylation or phosphorylation by Lck leading to its activation. The tyrosine phosphorylation sites in ZAP-70 include Y315, binding to the SH2 domain of Vav1 (Guanine nucleotide exchange factor for the GTPases Rac and CDC42) [19, 20], Y319, associating with SH2 domain of Lck [21, 22] and Y413 [23]. The activated kinase is then able to phosphorylate its substrates, such as adaptor protein LAT (Linker for Activation of T cells) [24, 25].
1.2.2.3. Adaptor proteins
Adaptors are proteins that do not have enzymatic or transcription-regulating activities but rather possess tyrosine residues as well as multiple protein protein interaction domains such as SH2 domains, SH3 domains, PH domain, PTB domains or proline rich regions. These structures enable adaptors to associate with other proteins and thereby allow the formation of multi-component signaling complexes. In T cells, specialized adaptor proteins, such as LAT and SLP-76 relay the signals from the TCR to intracellular signaling components.
LAT is a transmembrane protein with the molecular weight of 36-38 kDa. It is expressed in T lymphocytes, NK and mast cells, platelets, and pre-B cells. There are nine conserved tyrosine residues within the cytoplasmic tail of LAT. LAT is a substrate of ZAP-70 in response to T cell activation [25]. The sites of ZAP-70 phosphorylation in LAT have been mapped to the tyrosines 127, 132, 171, 191, and 226 [26]. Upon TCR activation, these tyrosines become phosphorylated and provide docking sites for multiple downstream signaling molecules including Grb2 (Growth factor Receptor-Bound protein 2), PLCγ1, and Gads (Grb2 related Adaptor Downstream of Shc) [26]. The phosphorylated LAT dependent interactions are essential to target these downstream molecules to the plasma membrane and in turn, to couple the TCR to downstream signaling cascades. A LAT deficient T cell line exhibits tyrosine phosphorylation defects including significantly diminished phosphorylation of PLCγ1, Vav, and SLP-76. These cells fail to flux intracellular calcium and cannot activate the Ras/Raf/ERK1/2 signaling cascade after TCR engagement [27, 28]. Re-expression of LAT reconstitutes all these events, demonstrating that LAT not only mediates TCR-induced tyrosine phosphorylation of many substrates but is also required for calcium flux, activation of Ras, and subsequent
downstream events leading to IL-2 gene expression. LAT also plays a crucial role in T cell development since LAT-deficient thymocytes are developmentally blocked at the DN stage [29].
Unlike LAT, SLP-76 is a cytoplasmic adaptor protein that contains an N-terminal domain with several tyrosine phosphorylation sites, a central proline-rich region, and a C-terminal SH2 domain [30, 31]. Tyrosine phosphorylation of SLP-76 at positions 113, 128, and 145 leads to its association with the SH2 domains of Vav, Nck (adaptor protein), and Itk (IL-2 inducible T cell Kinase) [32-35]. Mutation of these three tyrosines results in a severely diminished activation of downstream signaling cascades [36]. The proline-rich region of SLP-76 constitutively binds the SH3 domain of Gads, through which SLP-76 is targeted to phosphorylated LAT after TCR stimulation. The SH2 domain at the C-terminus of SLP-76 mediates its interaction with the cytosolic adaptor protein ADAP (Adhesion and Degranulation-promoting Adaptor Protein) [37] and the serine/threonine kinase HPK1 (Hematopoietic Progenitor Kinase 1) [38]. The critical function of SLP-76 in T cell activation has been demonstrated in cell lines and in vivo. In the SLP-76-deficient Jurkat T cell line J14, TCR activation fails to induce PLCγ1 phosphorylation, Ca2+ mobilization, ERK1/2 phosphorylation, and the activation of TCR-dependent transcription factors such as NF-AT and AP-1 [39]. In vivo, SLP-76 deficiency results in a complete arrest of T cell development at the DN3 stage [40, 41].
Figure 4. The binding partners of phosphorylated LAT after TCR stimulation.
LAT is a substrate of the protein tyrosine kinase ZAP-70 in T cells. Following phosphorylation at the sites of Y132, Y171, Y191, Y226, LAT recruits Grb2, Gads, and PLCγ1 via their SH2 domains.
1.2.2.4. PLCγ1 activation and calcium flux
The LAT/SLP-76 complex is important for plasma membrane recruitment and activation of PLCγ1 after TCR stimulation. The current model for the activation PLCγ1 postulates that in response to TCR stimulation, LAT is phosphorylated at Y132 which mediates an inducible interaction with the SH2 domain of PLCγ1. This interaction serves to recruit PLCγ1 to the plasma membrane. Phosphorylated LAT also associates with the Gads/SLP-76 complex, which in turn binds to Itk after TCR stimulation. The close proximity of Itk and PLCγ1 results in the phosphorylation and activation of PLCγ1 [42, 43]. Activated PLCγ1 hydrolyzes PIP2 (phosphatidylinositol 4,5-bisphosphate) producing two second messengers, DAG (diacylglycerol) and IP3 (inositol 1,4,5-trisphosphate) [44-46]. IP3 induces a transient increase in free intracellular Ca2+, which binds to calmodulin that, in turn, activates calcineurin, a Ca2+/calmodulin dependent protein phosphatase [47]. Activated calcineurin dephosphorylates NF-AT, thus allowing its translocation to the nucleus where the latter participates in IL-2 (Interleukin-2) transcription. Generation of DAG in response to TCR stimulation is also essential for the initiation of T cell activation. Proteins with a C1 domain, such as PKC (Protein Kinase C) [44, 48, 49] and RasGRP (Ras Guanyl nucleotide-Releasing Protein) [50], interact with DAG [51]. DAG is also required for the activation of PKD1 (Protein Kinase D1). Wood et al. have shown that the requirement of DAG for PKD1 activation integrates two DAG signals [52]. The first is Figure 5. The structure of SLP-76 and its binding partners important for T cell activation
SLP-76 is a cytoplasmic adaptor protein. It contains tyrosine phosphorylation sites (Y112, Y128,
Y145) at the N-terminal domain mediating its interaction with Vav, Nck, and Itk. The central
proline-rich domain is required for its constitutive interaction with Gads and PLCγ1, while the SH2 domain at the C-terminal is essential for the association with ADAP and HPK-1.
that DAG is required for PKC activation, which then colocalizes with PKD1 and phosphorylates serine 744 and 748 within PKD1. The second reflects a requirement for DAG binding to the CRD (Cysteine-Rich Domain) of PKD1, thereby relieving the inhibition of the catalytic domain of PKD1. PKD1 activity can be determined by analyzing S916 phosphorylation, which was previously reported to modify the conformation of the kinase and to influence the duration of its kinase activity [53].
1.2.2.5. Ras-Raf-ERK1/2 activation
PLCγ1 phosphorylation is also required for the activation of the Ras-Raf-ERK1/2 cascade. The GTPase Ras is a guanine nucleotide binding protein that cycles between an inactive GDP-bound and an active GTP-bound state. Ras activity can be regulated in a positive way by GEFs (Guanine nucleotide Exchange Factors) that promote the transition from the GDP-bound state to the active GTP-bound conformation and in a negative way by GAPs (GTPase-Activating Proteins), which stimulate Ras GTPase activity resulting in the hydrolysis of GTP to GDP and the accumulation of inactive Ras–GDP complexes. There are two GEFs proposed to activate Ras in T cells in response to TCR ligation. One is SOS (Son of Sevenless) which constitutively interacts with the adaptor protein Grb2 via binding of the SH3 domain in Grb2 to a proline rich region in SOS [54]. TCR cross-linking leads to LAT phosphorylation and its binding to Grb2, which correspondingly recruits SOS to the plasma membrane to trigger Ras activation. RasGRP is the other GEF in T cells. Recruitment of RasGRP to the membrane by DAG also leads to Ras activation [55]. GTP-bound Ras next recruits Raf-1 to the plasma membrane. There, Raf-1 is activated in a series of incompletely
Figure 6. The network of DAG-binding proteins.
Antigen receptor–coupled tyrosine kinases phosphorylate adaptors that trigger DAG (diacylglycerol) production via PLC-mediated hydrolysis of PtdIns(4,5)P2. DAG binds to conserved C1 domains
in many different molecules, including various isoforms of PKC (Protein Kinase C), PKD1 (Protein Kinase D1), Ras-GRP (activator of the GTPase Ras) and DGK (Diacylglycerol Kinase). Binding of DAG to C1 domains is essential in the regulation of protein localization but does not operate by a simple ‘on or off’ switch to stimulate the catalytic function of C1 domain– containing molecules. Instead, evidence is emerging that many DAG-binding proteins are substrates for and are regulated by PKC.
understood steps[56]. Activated Raf-1 then phosphorylates the cytosolic kinase MEK, which is responsible for the subsequent activation of the MAP kinase ERK1/2. Activated ERK1/2 translocates to the nucleus to target the ternary complex factor Elk-1, which is important for IL-2 production and T cell activation. Figure 7 represents some elements involved in TCR induced T cell activation.
1.2.3. The role of TCR signaling in thymocyte development
TCR signalling in mature T cells, as descirbed above, results in the induction of T cell effector functions, such as cytokine secretion and cell mediated cytoxicity. In the thymus, on the other hand, TCR-induced signals lead to positive or negative selection of DP thymocyte. Although it is widely accepted that the affinity of ligands dictates the outcome of thymocyte development, the molecular mechanisms involved are still
Figure 7. The TCR signaling pathway
Engagement of the TCR leads to the activation of Lck and ZAP-70 and in turn results in the phosphorylation of SLP-76 and LAT. Phosphorylated SLP-76 and LAT serve as docking elements for numerous cytoplasmic signaling molecules such as Grb2/SOS and PLCγ1. These molecules are involved in the activation of the Ras/MAP-kinase cascade, in the combination of calcium flux, in the activation of PKC and ultimately in the induction of trancriptional activity.
unknown. Initial studies have demonstrated that ERK1/2 plays an important role in positive selection [57, 58]. In ERK1-/- mice, thymocyte maturation beyond the CD4+CD8+ stage is reduced by half, with a similar diminution in the thymocyte subpopulation expressing high levels of TCR (CD3 high). However, more recent studies identified ERK1/2 as a potential point where the signals of positive and negative selection quantitatively diverge [57, 59]. The current model indicates that both the duration and the level of ERK1/2 activation influence negative selection as well as positive selection. Werlen et al. reported that weak but sustained activation of ERK1/2 occurs in response to low affinity ligands and results in positive selection. In contrast, negative selectors induce strong, but transient ERK1/2 activation [60]. In addition, Daniels et al. found that a small increase in ligand affinity for the TCR leads to a marked change in the activation and subcellular localization of Ras and ERK1/2 and the induction of negative selection [61].
Figure 8. TCR affinity model and the role of ERK1/2 activation during T cell development
The affinity model predicts that high affinity ligands trigger strong but transient ERK1/2 activation corresponding to negative selection whereas low affinity ligands induce a weak and sustained ERK1/2 activation correlating with positive selection.
1.2.4. Negative regulation of antigen receptor signaling in T cells 1.2.4.1. PTPases (Protein Tyrosine Phosphatases)
Since protein tyrosine phosphorylation plays an essential role in TCR signaling, removing the phosphorylation provides an easy way to inhibit T cell activation. It is generally accepted that PTPases are crucial for keeping T cells in a resting state in the absence of antigen. Several PTPases are reported to be capable of inhibiting T cell activation, such as Shp1 (SH2 domain containing PTP1) and CD148.
Shp1 is a cytoplasmic tyrosine phosphatase that is primarily expressed in hematopoietic cells [62]. Shp1 works as an important negative regulator of immune functions. The motheaten mice, which show point mutations within the Shp1 gene, develop a progressive inflammatory disease characterized by infiltrates of neutrophils and macrophages in the lung, skin, and other tissues [63]. Thymocytes and peripheral T cells from motheaten mice exhibit a markedly increased proliferative response to TCR stimulation compared to normal cells. Shp1 deficient thymocytes also show increased constitutive tyrosine phosphorylation of the TCR complex and enhanced and prolonged TCR-induced tyrosine phosphorylation of the ζ chain and CD3ε, as well as a number of cytosolic proteins [64]. Shp1 is reported to attenuate the earliest events in TCR signaling, since Lck and ZAP-70 have been identified as possible substrates for Shp1. Upon T cell activation, Shp1 binds to ZAP-70, resulting in an increase in Shp1 phosphatase activity and a decrease in ZAP-70 kinase activity [65, 66]. In addition, Stefanova et al. suggested that Shp1 can also help T cells to distinguish between high and low affinity ligands. Following TCR stimulation, activated Lck triggers Shp1 phosphorylation at Y564. This promotes Shp1 recruitment to the TCR via its binding to the Lck-SH2 domain, where Shp1 then dephosphorylates and inactivates Lck, thus terminating signals. However, high affinity ligands not only induce Shp1 phosphorylation, but also evoke ERK1/2-mediated Lck phosphorylation at S59, preventing Shp1 recruitment and thus Lck inactivation [66] (Figure 9).
CD148 is another protein tyrosine phosphatase that negatively regulates TCR signaling. CD148 was found to be weakly expressed on B and T cells, platelets, natural killer cells, certain dendritic cells as well as on mature thymocytes. CD148 is upregulated following in vitro activation of human peripheral T cells [67]. Overexpression of CD148 in Jurkat T cells inhibits TCR-induced NF-AT activation, ERK1/2 phosphorylation, calcium flux, PLCγ1 activation, and LAT phosphorylation [68]. Following stimulation with APCs loaded with staphylococcal enterotoxin superantigen (SAg), CD148 is excluded from the immunological synapse, sequestering it from its potential substrates. Once the T cell has disengaged from the APCs, CD148 can access
Figure 9. Shp1/Lck/ERK1/2-mediated feedback loops in TCR signaling.
(A) Upon TCR engagement with an agonist, Lck is activated, resulting in Shp1 phosphorylation at Y564 (pShp1) by Lck. This promotes pShp1 recruitment to the TCR via its binding to the
Lck-SH2 domain (pink), where pShp1 then dephosphorylates and inactivates Lck to terminate signaling. (B) If TCR signal strength exceeds a critical threshold (high affinity ligand), TCR-evoked ERK1/2 activation results in ERK1/2-mediated Lck phosphorylation at S59, preventing
Shp1 recruitment and thus Lck inactivation.
its substrates, such as LAT, PLCγ1, leading to their dephosphorylation, thereby downregulating T cell activation. Targeting the CD148 phosphatase domain to the immunological synapse potently inhibits NF-AT activation by TCR triggering [69]. 1.2.4.2. Negative regulation of T cell activation by ubiquitination and degradation TCR signaling can also be inhibited by removing critical mediators of activation through selective protein ubiquitination and degradation. Ubiquitination is a posttranscriptional modification of cellular proteins in which ubiquitin (Ub) monomers are either attached to lysine residues of target proteins or to Ub itself (to form poly-Ub chains). Attachment of a single ubiquitin motif to the substrate results in monoubiquitination while addition of ubiquitin chains to the substrate leads to the formation of polyubiquitination [70]. Two types of ubiquitin chains are abundant: K48 (lysine48 linked chain) and K63 (lysine63 linked chain). It has been suggested that K48 linked Ub chains serve as recognition signals for proteosomal degradation whereas K63 linked polyubiquitin chains regulate several cellular processes, such as DNA repair, signaling, endocytosis, vesicular trafficking, and cell-cycle progression [71]. The process of ubiquitination involves three enzymes: an Ub-activating enzyme (E1), an Ub-conjugating enzyme (E2), and an Ub ligase (E3) [72].
Cbl proteins are a family of E3 ligases of three related gene products c-Cbl, Cbl-b, and Cbl-c. All three Cbls have an N-terminal TKB (Tyrosine Kinase Binding) domain which mediates the binding to specific phosphotyrosine motifs on target molecules and a Ring finger domain that interacts with the E2 conjugase and position it so that the ubiquitin moiety can be transferred from E2 conjugase to the substrate via the E3 ligase (Figure 10). c-Cbl and Cbl-b have additional C-terminal tails composed of proline rich regions that contain functional SH3 binding sites and tyrosine residues that enable Cbl to interact with the SH2 domains of other proteins [73]. Cbl appears to be essential for the negative regulation of TCR signaling, since T cells from c-Cbl/Cbl-b double knockout mice are hyperresponsive towards TCR stimulation. This is partly due to the fact that TCR internalization into the lysosomes was blocked in the double knockout T cells [74].
1.3. T cell apoptosis
In response to antigens, which are processed and presented to T cells by APCs, antigen specific T cells begin to proliferate and differentiate. This process is essential for the conversion of naïve T cells into effector T cells, whose function is to eliminate invading microbes. The induction of apoptosis is another central issue during T cell activation. Apoptosis is responsible for maintaining homeostasis within the immune system, since unlimited proliferation of lymphocytes results in autoimmunity. Apoptosis involves a series of biochemical reactions leading to a variety of morphological changes including DNA cleavage, nuclear condensation, and fragmentation. It is also accompanied by surface exposure of phosphatidylserine, shrinking and blebbing of the plasma membrane, and formation of apoptotic bodies containing the condensed cell organelles and chromatin fragments. Caspases, a family of cysteinyl proteases, play an essential role in the regulation and execution of apoptosis [75, 76]. Caspases are synthesized as inactive enzyme precursors with a prodomain of variable length followed by a large subunit of 20 kDa (p20) and a small subunit of about 10 kDa (p10). The inactive caspases must undergo processing and activation before they can mediate apoptosis. The mechanism for procaspase activation involves procaspase cleavage at a specific asp-X bond resulting in the formation of the active caspase as a tetramer of two p20-p10 heterodimers and the release of the prodomain [77]. Among these caspases, caspase-3 serves as a downstream “apoptosis executor”. Its activation is involved in both the intrinsic and extrinsic apoptosis pathways. Caspase-3-defective peripheral T cells are less susceptible to CD3ε- and Fas
Figure 10. Model of Cbl Ubiquitin Ligase Function
An ubiquitin-activating enzyme (E1, purple) activates Ub and transfers it to an ubiquitin-conjugating enzyme (E2, yellow) which interacts with a ubiquitin ligase (E3) and transfers Ub to the target protein (peach). The RING finger of the E3 ligase mediates its binding to the E2 conjugase. Multiple motifs in Cbl proteins, such as TKB (pink) domain, the proline-rich region (blue), or the phosphorylated tyrosine residues (not shown) serve to recruit the substrates for ubiquitinylation. The induced ubiquitinylation may be in the form of monoubiquitin units or polyubiquitin chains, leading to lysosomal or proteasomal targeting, respectively.
receptor induced apoptosis and activation-induced cell death than similar cells from wild-type mice [78].
1.3.1. Two signaling pathways leading to apoptosis
T cells have two different apoptotic pathways which are mediated by distinct initiator caspases but converge into the same executor, caspase-3 (Figure 12). The first pathway also called the “extrinsic” apoptosis signaling pathway is mediated by death receptors [79, 80]. The death receptors are transmembrane proteins. Death domains, which are responsible for apoptotic signal transduction, are located in the cytoplasmic tail of the receptor. Six members of this subfamily are known so far [81]. In T cells only TNF-receptor and FasR (also known as CD95) are essential for T cell apoptosis [82, 83]. The ligation of death receptors by their ligand causes the formation of DISC (Death-Inducing Signaling Complex), which involves the oligomerized death receptors and their death domain containing adaptor FADD, which is recruited to the cytoplasmic tail of the death receptor. Through the homotypic interaction of DEDs (Death Effector Domains), FADD can further recruit procaspase-8 to the DISC. Upon recruitment, caspase-8 undergoes processing by cleavage and forming dimers [84]. Once activated, caspase-8 will induce the activation of caspase-3 and T cell apoptosis.
Figure 11. Mechanism of caspase activation
Procaspase contains a small amino-terminal prodomain, a p20 and a p10 subunit. The interfaces of these domains contain critical aspartate residues that are caspase cleavage sites. These are cleaved by active caspases and form p10−p20 heterodimers in a tetrameric structure containing two active caspase sites.
In the other pathway, also called the “intrinsic” apoptosis pathway, apoptosis is initiated through regulating mitochondrial membrane integrity. The presence of apoptotic stimuli leads to the disruption of the mitochondrial membrane and the leakage of the pro-apoptotic molecule cytochrome C into the cytosol. In the presence of ATP, cytochrome C can bind Apaf-1 (Apoptosome scaffold Protein Apoptotic protease-activating Factor 1), which further results in the activation of caspase-9 [85, 86]. The events leading to the disruption of the mitochondrial membrane and release of pro-apoptotic molecules are not clear. However, the Bcl-2 family proteins represent checkpoint that regulate the integrity of the mitochondrial membrane. The Bcl-2 family consists of two groups of molecules that are either pro-apoptotic or anti-apoptotic. Anti-apoptotic molecules, such as Bcl-2 and Bcl-xL [87] are characterized by four Figure 12. Illustration of apoptosis driven by death receptors or Bcl-2 family members in activated T cells.
In type I apoptosis oligomerization of death receptors by their ligands induces recruitment of adaptor proteins such as FADD. These adaptor proteins bind to the cytoplasmic tail of receptors through homologous DD interactions. Procaspase-8 is then recruited into the complex and activated. Once activated, caspase-8 cleaves and activates caspase-3, which in turn cleaves other caspases, thus leading to apoptosis. In type II apoptosis, high levels of BAD, BID, and BIM by unknown mechanism signal BAX or BAK to form pores in mitochondria and cause the release of cytochrome C from mitochondria. Once released, cytochrome C interacts with procaspase-9. This complex favors the activation of caspase-9 which, when activated, cleaves and activates caspase-3, thus leading to apoptosis. The effects of pro-apoptotic molecules can be blocked by anti-apoptotic molecules, such as Bcl-2 or Bcl-xL.
conserved BH domains in addition to a C-terminal hydrophobic transmembrane domain through which the anti-apoptotic molecules are localized in the membranes of the mitochondria, the endoplasmic reticulum (ER), and the nucleus. Anti-apoptotic molecules are supposed to protect the cell from apoptosis. The pro-apoptotic group includes molecules like Bid, Bim, Bak, and Bax [88-91]. In resting cells, the pro-apoptotic molecules are located in the cytosol or loosely attached to the endoplasmic reticulum membrane. In response to apoptotic stimuli, they translocate to the mitochondria, where they oligomerize, integrate into the membrane and form holes in the mitochondrial membrane, thus inducing the release of apoptogenic proteins. The anti-apoptotic molecules prevent the oligomerization and insertion of some pro-apoptotic molecules into the mitochondrial membrane. Of note the BH3-protein Bid is one cirtical determinant of a crosstalk between the intrinsic and the extrinsic apoptotic pathway [92].
1.4 Aims of the present research
Engagement of the TCR can induce different functional outcomes such as differentiation, survival, or apoptosis. However, despite the progress in our understanding of the signaling pathways regulating T cell function, howthe TCR can transmit signals leading to distinct cellular responses is so far not completely understood. Thus, to shed light on this issue, I firstly identify ligands that induce two different cellular outcomes, namely proliferation or apoptosis. Successively, I investigated signaling pathways triggered by ligands inducing proliferation or apoptosis, by using antibodies raised against phosphorylated sites. An increasing body of evidences indicates that both the amplitude and the duration of signaling events can regulate cellular responses. For example, EGF transiently activates ERK1/2, and thus stimulates cell proliferation, whereas NGF stimulation leads to sustained ERK1/2, resulting in neuronal differentiation. Thus, I subsequently address the activation kinetics of signaling molecules under proliferation versus apoptosis condition. Finally, it is known that the subcellullar localization of signaling molecules could also regulate cellular outcomes. Therefore, I also examined the subcellular localization of molecules mediating the TCR induced ERK1/2 activation.
2. Materials and methods
2.1. Materials 2.1.1. Instruments
AutoMACS Separator (Miltenyi Biotec)
ELISA reader: Expert Plus Microplate Reader (ASYS) Pipette (Eppendorf)
Power PAC 200 (Bio-Rad) Nova Blot (Amersham)
Centrifuge 5415R (Eppendorf)
Multifuge 1 S-R Centrifuge (Heraeus) Incubator (Heraeus)
Kodak Image Station 2000R (Kodak)
Leica TCS-NT laser-scanning confocal microscope (Leica Microsystems) LSR flow cytometer (BD Bioscience)
FACS Calibur flow cytometer (BD Bioscience) PCR machine (Bio-Rad)
Pipette boy (Eppendorf)
Scintillation counter (1450 MicroBeta Trilux; PerkinElmer) Thermomixer compact (Eppendorf)
Documentation station (Herolab GmbH) Whell shaker Duomax 1030 (Heidolph) NeoLab Rotator 2-1175 (NeoLab) Bio-Rad Mini DNA system (Bio-Rad) Bio-Rad Mini protein system (Bio-Rad) Neubauer counting chamber (Marienfeld)
Coverslip 24x50 mm (for immunofluorescence) (Roth) Coverslip (for Neubauer counting chamber) (Roth)
12 spot slide (precoated with poly-L-Lysine) (Marienfeld) 2.1.2. Plastic ware
Eppendorf tubes (Eppendorf) PCR soft tubes (Biozym) FACS tubes (Falcon)
Plastic pipettes (5 ml, 10 ml, 25 ml, 50 ml) (Costar) Eppendorf tips (10 µl, 200 µl, 1 ml) (Eppendorf) Syringes (2 ml, 20 ml) (BD Bioscience)
15 ml and 50 ml tubes (Greiner) 96 well cell culture plates (TPP)
96 well cell culture plates (U bottom) (Costar) 48 well cell culture plates (Nunclon)
40 μm diameter pore-size strainers (BD Bioscience) 2.1.3. Kits
Nucleospin Tissue kit (Macherey-Nagel, # 740 952.250)
Rh Annexin V/FITC kit (Bender MedSystems, # bms306FICE) Caspase-3 detection kit (Calbiochem, # QIA91)
Pan T cell isolation kit, mouse (Miltenyi Biotec, # 130-096-861) CD8 T cell isolation kit, mouse (Miltenyi Biotec, # 130-090-859) Taq-polymerase kit (PeqLab, # 01-1030)
ECL Western Blotting detection reagent (Amersham, # 2106) 2.1.4. Chemical Reagents
30% Acrylamide/BIS, 37.5:1 (Bio-Rad, # 161-0158) Acetic acid (Roth, # 3738.1)
Agarose (Peqgold universal agarose) (PeqLab, # 35-1020) APS (Ammoniumpersulfat) (Roth, # 9592.3)
2-mercaptoethanol (Sigma, # M7154) Bromphenolblue (Roth, # A512.1)
BSA (Bovine Serum Albumin) (Sigma, # A9647) dNTP 25 mM (Fermentas, # 0161)
DMSO (Dimethyl Sulfoxide) (Roth, # A994.2)
EDTA (Ethylenediaminetetraacetic acid) (Sigma, # E5134) Ethanol, 99% (Roth, # 9065.1)
Ethidium bromide, 10 mg/ml (Roth, # 2218.1)
Fetal Bovine Serum (FBS) (Pan Biotech GmbH, # P30-3302) GeneRuler 50 bp DNA Ladder (Fermentas, # SM0371) Glycerol (Sigma, # G6279)
Glycine (Roth, # 3908.2)
Horse serum (Biochrom AG, # 9133) Indo-1 (Invitrogen, # I1223)
Ionomycin (Sigma, # I3909)
Igepal CA-630 (Nonidet P-40 (NP-40)) (Sigma, # I3021)
LM (lauryl maltoside / n-Dodycyl-β-D-maltoside) (Calbiochem, # 324355) Methanol, 99.9% (Roth, # 4627.1)
Milk powder (Roth, # T145.2)
Mowiol 4-88 (Calbiochem, # 475904) NaF (Sodium Fluoride) (Sigma, S-7920) NaN3 (Sodium Azide) (Roth, # K305.02) NaCl (Sodium Chloride) (Roth, # 3957.2) NH4Cl (Ammonium Chloride) (Roth, # A4514)
Pageruler prestained protein ladder (Fermentas, # SM 0671) PFA (Paraformaldehyde) (Merke, # 1.04005.1000)
PBS (Phosphate Buffered Saline) (Biochrome, # L1825)
Penicillin/Streptomycin 10000U/10000 µg/ml (Biochrom AG, # A2213) Phalloidin-TRITC (Tetramethylrhodamine B isothiocyanate) (Sigma, # P1951) PMA (Phorbol Myristate Acetate) (Sigma, # P8139)
PMSF (Phenylmethylsulfonyl Fluoride) (Roth, # 6367.1) Ponceau S (Sigma, # P-3504)
Protein-A agarose beads (Santa Cruz, # sc-2001) Roti Nanoquant reagent (Roth, # K880.1)
RPMI 1640 medium (Biochrome, # FG1215)
RPMI 1640 medium without phenol red (Invitrogen, # 11835) SDS (Sodium Dodecyl Sulfate) (Roth, # 2326.2)
Streptavidin (Dianova, # 016-000-113)
TEMED (Tetramethylethylenediamine) (Roth, # 2367.3) [3H]-Thymidine (MP Biomedicals, # 24043)
Tris (Tris (hydroxymethyl)-aminomethan) (Roth, # 4855.2) Triton X-100 (Sigma, # T9284)
Trypan blue solution 0.4% (Sigma, # T8154) Tween 20 (Roth, # 9127.2)
2.1.5. Antibodies
2.1.5.1. Antibodies for stimulation
Biotin-conjugated anti-mouse CD3ε monoclonal antibody (mAb) (hamster) (BD Bioscience, # 553060)
Biotin-conjugated anti-mouse CD8α mAb (rat) (BD Bioscience, # 553029) Biotin-conjugated anti-mouse CD8β mAb (rat) (BD Bioscience, # 553039)
Biotin-conjugated anti-mouse TCR β chain mAb (hamster) (BD Bioscience, # 553168) 2.1.5.2. Antibodies for flow cytometric analysis
FITC (Fluorescein-5-isothiocyanate)-conjugated anti-mouse Vα2 T-cell receptor mAb (mouse) (BD Bioscience, # 553288)
FITC-conjugated anti-mouse CD8α mAb (rat) (BD Bioscience, # 553031)
FITC-conjugated anti-Fas ligand mAb (mouse) (Kamiya Biomedical Co, # MC-136) FITC-conjugated anti-Fas receptor mAb (hamster) (BD Bioscience, # 15404D) FITC-conjugated anti-mouse LAMP1 mAb (rat) (BD Bioscience, # 553793) FITC-conjugated anti-mouse CD69 mAb (mouse) (BD Bioscience, # 553236)
PE (Phycoerythrin)-conjugated anti-mouse CD3ε mAb (hamster) (BD Bioscience, # 553063)
PE-conjugated anti-mouse CD25 mAb (rat) (BD Bioscience, # 553866) PE-conjugated anti-mouse CD4 mAb (rat) (BD Bioscience, # 553049)
Cy5 (Cyanine 5)-conjugated anti-mouse CD4 mAb (rat) (BD Bioscience, # 553050) 2.1.5.3. Antibodies for Western blotting and Immunoprecipitation (IP)
Anti-phospho-ZAP-70 (Tyr 319) /Syk (Tyr 352) antibody (rabbit) (Cell Signaling, # 2701)
Anti-phospho-PLCγ1 (Tyr 783) antibody (rabbit) (Santa Cruz, # 12943) Anti-phospho-LAT (Tyr 171) antibody (rabbit) (Cell Signaling, # 3581) Anti-phospho-PKD (Ser 916) antibody (rabbit) (Cell Signaling, # 2054)
Anti-phospho-p44/p42 MAP kinase (Thr 202/Tyr 204) antibody (rabbit) (Cell Signaling, # 9101)
Anti-phospho-PKB (Ser 473) mAb (rabbit) (Cell Signaling, # 9271) Anti-ubiquitin mAb (mouse) (Cell Signaling, # 3936)
Anti-ZAP-70 mAb (mouse) (BD Bioscience, # 610239) Anti-ERK1/2 antibody (rabbit) (Cell Signaling, # 9102)
Anti-LAT mAb (mouse) (BD Bioscience, # 611108) Anti-PKB antibody (rabbit) (Cell Signaling, # 9272) Anti-PLCγ1 antibody (rabbit) (Santa Cruz, # sc-81) Anti-Bcl-xL antibody (rabbit) (BD Bioscience, # 556361) Anti-CD107a (LAMP-1) mAb (rat) (BD Bioscience, # 553792) Anti-Bim antibody (rabbit) (BD Bioscience, # 559685)
Anti-CD3ζ mAb (mouse) (Santa Cruz, # sc-1239) Anti-β-actin mAb (mouse) (Sigma, # A1978)
Anti-Rab5 mAb (mouse) (BD Bioscience, # 610281) Anti-Ras mAb (mouse) (Oncogene, # AB-3)
2.1.5.4. Other antibodies
Anti-IL-2 mAb (rat) (BD Bioscience, # 554375) 2.1.5.5. Secondary antibodies
Peroxidase-conjugated affinipure goat anti-mouse IgG+IgM (H+L) (Jackson Immunoresearch, #115-035-068)
Peroxidase-conjugated affinipure goat anti-rabbit IgG (H+L) (Jackson Immunoresearch, # 115-035-045)
Peroxidase-conjugated affinipure goat anti-rat IgG (H+L) (Jackson Immunoresearch, # 112-035-167)
FITC-conjugated affinipure donkey anti-rabbit IgG (H+L) (Jackson Immunoresearch, # 711-095-152)
FITC-conjugated affinipure goat anti-rat IgG (H+L) (Jackson Immunoresearch, # 112-095-167)
FITC-conjugated affinipure donkey anti-mouse IgG (H+L) (Jackson Immunoresearch, # 715-095-150)
Cy3 (Cyanine3)-conjugated affinipure goat anti-rat IgG (H+L) (Jackson Immunoresearch, # 112-165-167)
Cy3-conjugated affinipure goat anti-rabbit IgG (H+L) (Jackson Immunoresearch, # 111-165-144)
Cy3-conjugated affinipure donkey anti-mouse IgG (H+L) (Jackson Immunoresearch, # 715-165-151)
Cy5-conjugated affinipure donkey anti-mouse IgG (H+L) (Jackson Immunoresearch, # 715-175-151)
2.1.6. Streptamers
MHC-I-Strep H-2Kb Ovalbumin (IBA, # 6-7015-001) Strep-Tactin (IBA)
Strep-Tactin PE (IBA, # 6-5000-001)
Strep-Tactin APC (Allophyocyanin) (IBA, # 6-5000-002) 2.1.7. Mouse strains
OT1-TCR transgenic (tg) mice were kindly provided by Dr. Percy Knolle (Institute of Molecular Medicine and Experimental Immunology, University of Bonn, Germany). Perforin knock-out (perforin-/-) mice were kindly provided by Dr. Andreas Ambach (Department of Dermatology and Venerology, Otto-von-Guericke-University, Germany).
2.2. Methods
2.2.1. Animal experimentation 2.2.1.1. Mice condition and handling
Mice were kept in the central animal facility at the Otto-von-Guericke-University of Magdeburg and maintained in pathogen-free conditions. All experiments involving mice were performed according to the guidelines of the State of Sachsen-Anhalt, Germany.
2.2.1.2. Genomic DNA isolation from mouse tails
Genomic DNA was purified according to the manufacturer’s protocol. Briefly, a 1 cm piece of mouse tail was provided from the animal facility and placed into a 1.5 ml eppendorf tube. The mouse tail was pre-digested by incubation with 180 µl buffer T1 (cell lysis solution) and 25 µl proteinase K solution at 56°C for 3-5 h. The mouse tail was further digested by incubating with 200 µl of lysis buffer for 10 min at 70°C, under constant shaking. The condition for DNA binding to the silica membrane was adjusted by adding 210 µl 99% ethanol and vortexing vigorously. The lysis solution was added next onto the column and centrifuged for 1 min at 11,000 g leading to DNA
binding to the silica membrane. The flow-through was discarded and the column was placed back into the collecting tube. The silica membrane was washed with 500 µl buffer BW and 600 µl buffer B5, respectively. The silica membrane was dried by centrifugation for 1 min at 11,000 g. DNA was eluted from the silica membrane with 100 µl prewarmed elution buffer BE (70°C) and centrifuged at 11,000 g for 1 min. 2.2.1.3. Genotyping of OT1-transgenic mice strain by PCR (Polymerase-Chain Reaction)
Genotyping of the OT1-transgenic mice was performed according to the PCR protocol established by Jackson Laboratories.
Primers used for genotyping (Metabion):
Primer 1 (internal control F): 5’- CAA ATG TTG CTT GTC TGG TG -3’ Primer 2 (internal control R): 5’- GTC AGT CGA GTG CAC AGT TT-3’ Primer 3 (OT1-TCR F): 5’- AAG GTG GAG AGA GAC AAA GGA TTC-3’ Primer 4 (OT1-TCR R): 5’- TTG AGA GCT GTC TCC-3’
The following reaction mix was pipetted into the PCR tube according to the manufacturer’s protocol. Primer 1 0.25 µl (1 µM) Primer 2 0.25 µl (1 µM) Primer 3 0.125 µl (0.5 µM) Primer 4 0.125 µl (0.5 µM) Taq polymerase 0.5 µl (2.5 U) 5 x enhancing buffer 5 µl 10 x reaction buffer 2.5 µl dNTP (mix) 0.25 µl (200 µM) dH2O up to 23.5 µl
To each reaction 1.5 µl DNA was added.
The following program was run in a PCR machine:
Hot start 94°C, 3 min
Denaturation 94°C, 30 sec
Hybridization 52°C, 30 sec 38 cycles
Final elongation 72°C, 2 min 2.2.1.4. Gel electrophoresis of nucleic acids
1 x TAE buffer: 10 mM Tris 0.1142% acetic acid 1 mM EDTA pH 8.0
Agarose gel 2%: 2 g agarose
100 ml 1 x TAE buffer
10 µl ethidium bromide (10 mg/ml)
6 x loading buffer: 30% glycerol
20 mM Tris (pH 7.6)
2 mM EDTA
0.02% bromphenolblue
0.02% xylenxylanol
Marker: GeneRuler 50 bp DNA Ladder
The reaction was analyzed by mixing the PCR products with 6 x loading buffer and loaded on a 2% agarose gel supplemented with ethidium bromide. Electrophoresis was carried out with a Bio-Rad Mini DNA system in TAE-buffer at 100 V for 30 min. DNA fragments were visulized by UV light and picture was taken by the documentation station.
2.2.2. T cell purification
2.2.2.1. Preparation of single cell suspension from mouse spleen
Mice were sacrificed and the spleens were removed. To disrupt the organs and to release the cells, the spleen was mashed through a 40 μm diameter pore-size strainer with a syringe plunger at RT (Room Temperature). Cells were collected in a 15 ml tube and washed by adding ice-cold PBS to a final volumn of 15 ml and centrifuged for 10 min, 370 g, at 4°C. The pellet was resuspended in 10 ml ice-cold PBS and the cell number was determined.
2.2.2.2. Cell counting
Trypan blue solution: 0.4% in PBS
Trypan blue solution was first diluted to 0.1% with PBS. The cell number was determined using a Neubauer counting chamber. An aliquot of the cell suspension was mixed with an equal volume of 0.1% trypan blue solution in PBS. Cell solution was pipetted into the Neubauer counting chamber. Cells in 1 x 10-4 ml were counted and the concentration of cells was determined using the following formula:
Negative trypan blue cells in one quadrant x 2 x dilution factor x 104= cells /ml. Mean values of four quadrants were always calculated.
2.2.2.3. Purification of CD8+ T cell
Because OT1-TCR tg mice contain only CD8 T cells, a Pan T cell isolation kit was used to purify these cells. The single cell suspension described in 2.2.2.1 was centrifuged at 370 g for 10 min. The cell pellet was resuspended in ice-cold PBS at a concentration of 10 x 106 cells/45 µl. Staining was performed with 5 µl Biotin-Antibody Cocktail (cocktail of biotin-conjugated monoclonal antibodies against CD11b (Mac-1), CD45R (B220), DX5 and Ter-119) and incubated for 10 min on ice. Subsequently, the cell suspension was mixed together with 40 µl of ice-cold PBS per 10 x 106 cells and 10 µl of anti-Biotin MicroBeads. The mixture was incubated for 15 min on ice. The cells were next washed once with 10 ml of ice-cold PBS and centrifuged at 370 g for 10 min. The supernatant was removed and the cell pellet was resuspended in PBS at the concentration of 1 x 108 cells/ml. The magnetic separation was performed by using the Auto-MACS separator. The purity of isolated cells was determined by flow cytometry.
Since perforin-/- mice were not crossed onto the OT1-transgenic background, a CD8+ T cell isolation kit was used to isolate CD8+ T cells from perforin-/- mice. The purification procedure was the same as described above.
2.2.3. T cell stimulation in vitro
2.2.3.1. T cell stimulation in vitro with OT1-streptamers Mouse medium: RPMI-1640
10% FBS
1% antibiotics (penicillin and streptomycin) 1.75 µl 2-mercaptoethanol (in 500 ml RPMI)
Prior to stimulation of T cells with streptamers, 0.75 µg Strep-Tactin was incubated with recombinant monomeric biotinylated-MHC-I (1 µg) in a final volume of 50 µl with PBS or mouse medium at 4°C for 45 min according to the manufacturer’s instructions. Recombinant monomeric biotinylated H-2Kb molecules presenting the ovalbumin SIINFEKL peptide for the OT1–TCR were used in this study. 2 x 106 purified T cells were resuspended in 50 µl PBS or mouse medium. Subsequently, the cell solution was mixed with the OT1-streptamers (pMHC-strep-tactin-PE) at 37°C for the indicated time points. Stimulation was stopped by adding 1 ml ice-cold PBS. The procedure was continued according to the specific purpose.
2.2.3.2. CD3/CD8 mAbs stimulation
2 x 106 purified T cells were resuspended in 100 µl of PBS or mouse medium. Before stimulation, the cells were pre-incubated with biotinylated CD3ε mAb and biotinylated CD8 mAb (both ranging from 10 µg/ml to 1 µg/ml) at 37°C for 1 min. Stimulation started when cells were crosslinked with 50 µg/ml streptavidin. 1 ml of ice-cold PBS was added to stop the reaction. The procedure was continued according to the specific purpose.
2.2.3.3. Plate-bound CD3/CD8 mAbs stimulation
Fourty-eight well plates were coated with 10 µg/ml of CD3/CD8 mAbs, overnight at 4°C. Subsequently, the plates were washed three times with PBS. Purified T cells were cultured in mouse medium at the concentration of 2 x 106/300 µl. Cell suspension was added to the antibody-coated plate and maintained in culture for the indicated time at 37°C and 5% CO2. The stimulation was stopped by adding 1 ml ice-cold PBS. The procedure was continued according to the specific purpose.
2.2.4. Surface staining and FACS (Fluorescence Activated Cell Sorting) analysis 2.2.4.1. Extracellular staining
1 x 106 cells were centrifuged for 5 min, 400 g, at 4°C. The pellet was resuspended in 100 μl of the indicated antibody solution. After 30 min of incubation in the dark at 4°C, the cells were washed with PBS. The cell pellet was resuspended in 300 μl PBS and measured by the FACS Calibur machine. Data were analyzed using the CellQuest software.
2.2.4.2. TCR internalization assay
Purified T cells were left untreated or stimulated with CD3/CD8 mAbs or OT1-streptamers for 30 min, 1 h and 2 h at 37°C. Cells were then incubated with FITC- conjugated Vα2 antibody for 15 min at 4°C in the dark. The expression of TCR-Vα2 on the cell surface was analyzed by flow cytometry.
2.2.5. Proliferation assay
Purified T cells were cultured in mouse medium using U-bottomed 96-well plates at a concentration of 2.5 x 104 cells/well. Cells were left either untreated or stimulated with OT1-streptamers (1 µg), CD3/CD8α (10 µg/ml), CD3/CD8β (10 µg/ml), CD3/CD8α/CD8β (10 µg/ml), TCR/CD8α (10 µg/ml), and PMA (40 ng/ml)/ionomycin (100 ng/ml) for 72 h at 37°C. Cells were then labeled with [3H]-thymidine (0.3 µCi/well from ICN) for 8 h, harvested onto glass fiber filter and counted with a scintillation counter.
2.2.6. Apoptosis assay
Cell apoptosis was analyzed by the ability of annexin V to bind to exposed phosphatidylserine residues at the outer leaflet of the plasma membrane in combination with the application of PI (Propidium Iodide). Purified T cells were resuspended in mouse medium at the concentration of 1 x 106 cells/ml in a 48-well cell culture plate. Cells were left untreated or treated with OT1-streptamers (1 µg) or CD3/CD8α (10 µg/ml) at 37°C for 8 h and 24 h, respectively. Cells were next harvested and washed with PBS. The cell pellet was resuspended in 195 µl of 1x binding buffer. The staining was performed by mixing the cell solution with 5 µl of annexin V-FITC and incubating for 10 min at RT in the dark. The cells were washed once with PBS and resuspended in 190 µl 1x binding buffer together with 10 µl PI. Samples were analyzed by flow cytometry within one hour. The apoptosis analysis was performed according to the protocol established by Bender MedSystems.
2.2.7. Caspase-3 activity assay
To detect activated caspase-3 in living cells, the caspase-3 detection kit was used. Caspase-3 activity was measured using FITC-DEVD-FMK, which is a cell-permeable, non-toxic inhibitor that binds irreversibly to the activated caspase-3 in apoptotic cells. T cells were resuspended in mouse medium at the density of 1 x 106 cells/ml and left untreated or treated with 10 µg/ml CD3/CD8 mAbs or 1 µg OT1-streptamers, for 8 h
and 24 h at 37°C. Cells were then transferred into a FACS tube, washed with PBS and resuspended in 1 ml PBS. Caspase-3 activity was assessed by incubation wih 1 µl of FITC-DEVD-FMK for 45 min at 37°C in the dark. Cell were washed once with PBS and resuspended in 300 µl PBS. Caspase-3 activity was assessed by flow cytometry. 2.2.8. Calcium flux determination
Calcium flux measurements were performed by measuring the ratio of the fluorescent signals given by staining with the Ca2+-binding fluorochrome Indo-1AM to either saturated or free Ca2+ molecules. CD8+ T cells (2 x 107 cells/ml) were resuspended in RPMI-1640 medium (phenol-red free; supplemented with 10% FCS) and loaded with 3.75 µg/ml Indo-1-AM at 37°C for 45 min. After washing, the cells were incubated in the same medium at 37°C for additionally 45 min followed by centrifuging for 10min, 400 g. Cell pellets were resuspended in the same medium at the concentration of 2x106 /100 µl. Cell suspension was incubated either with CD3/CD8 mAbs (each 10 µg/ml) at 37°C for 2 min to establish the baseline and cross-linked with streptavidin (50 µg/ml) or with OT1-streptamers (1 µg/reaction). As a cell-autonomous Ca2+“charge” control, full-scale deflection of the calcium flux was measured by addition of ionomycin (10 µg/ml). Data for calcium mobilizationwere acquired on an LSR flow cytometer and ratiometric analysiswas performed using the Flow Jo software.
2.2.9. Immunoblotting 2.2.9.1. Cell lysis Lysis buffer: 1% LM 1% NP-40 1 mM Na-monovanadate 1 mM PMSF 50 mM Tris-HCl (pH 7.4) 10 mM NaF 10 mM EDTA 0.16 M NaCl
After stimulation, 2x106 cells were washed once with ice-cold PBS. Cells were resuspended in 40 µl lysis buffer and incubated for 20 min on ice. Samples were then centrifuged for 10 min at 16,000 g, 4°C and the post-nuclear supernatant was transferred into a new eppendorf tube.
2.2.9.2. Protein concentration measurement BSA standard (0-100 μg/ml)
Roti-Nanoquant
Protein concentrations were determined by using the Bradford protein assay. According to the manufacturer’s protocol, a working solution was prepared by diluting the 5x Roti-Nanoquant to 1x with dH2O. Samples were pre-diluted with dH2O. 50 μl of the prediluted samples were then transferred to a 96-well plate and incubated with 200 μl of 1x Roti-Nanoquant. Absorption was measured using an ELISA reader at 570 nm. BSA (0-100 μg/ml) was used as standard and the protein concentration was calculated on the basis of the derived standard curve.
2.2.9.3. SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) SDS is an anionic detergent which denatures secondary and non–disulfide–linked tertiary structures and applies a negative charge to proteins in proportion to their mass. In SDS-PAGE, the migration of proteins is related to their molecular weight.
10% and 12% SDS-PAGE separating gel: 4.2 ml (10%) or 3.44 ml (12%) dH2O 2.46 ml 1.5 M Tris-HCl, pH 8.8 3.2 ml (10%) or 3.94 ml (12%) 30% acrylamide/BIS 0.1 ml 10% SDS 0.05 ml 10% APS 5 µl TEMED
SDS-PAGE stacking gel: 2.4 ml dH2O
0.5 ml 30% acrylamide/BIS 1 ml 0.5 M Tris-HCl pH 6.8 0.04 ml 10% SDS
0.04 ml 10% APS 0.004 ml TEMED
SDS-PAGE running buffer (1x): 25 mM Tris, 250 mM glycine, 0.1% SDS
5x reducing loading buffer : 50% glycerol
330 mM Tris, pH 6.8 10% SDS
0.01% bromphenolblue 10% 2-mercaptoethanol
Marker: pageruler prestained protein ladder
Cell lysates and 5x sample buffer were mixed and boiled at 99°C for 5 min. The samples were then loaded and resolved on a 10% or 12% SDS-PAGE gel. For each lane, 20 µg of total protein were loaded. Electrophoresis was conducted with Bio-Rad Protein system. Gels were run at 120 V for 90 min.
2.2.9.4. Western blotting analysis and immunoblotting
Protein transfer buffer (1x): 39 mM glycine 48 mM Tris
0.037% SDS 20% methanol
TBS 0.01M Tris
0.15M NaCl
Blocking buffer: 5% milk powder in TBS
Washing buffer: 0.02% Tween 20 in TBS
Ponceau S solution: 0.1% Ponceau S in 5% acetic acid
HRP inactivation buffer: 1% NaN3 in PBS
Stripping buffer: 0.7% 2-mercaptoethanol 2% SDS
The proteins seperated by gel electrophoresis were then transferred to a nitrocellulose membrane, where they were detected using antibodies specific to the target proteins. In some experiments, equal loading was controlled by incubation of the membrane with Ponceau S solution for 2 min. The staining was removed by washing with TBS. Blocking of the membrane was performed using blocking buffer for 30 min to prevent unspecific binding to the membrane. Subsequently, membranes were probed with a primary antibody for 1 h at RT. After washing 3 times with washing buffer, the membrane was incubated with the appropriate peroxidase-conjugated secondary antibody for 1 h at RT. The membrane was further washed 3 times with washing buffer and the bound antibodies were then visualized using an ECL (Enhanced Chemiluminescence) detection system according to the manufacturer’s instructions. The intensity of the detectedbands was acquired using the Kodak Image station 2000R and analysiswas performed using ID Image software (Kodak). The membranes can be stripped and reprobed a number of times by incubating with stripping buffer for 20 min at 50°C. The stripping buffer was used to remove the bound primary and secondary antibodies from the membrane. Prior to the addition of the next primary antibody, the membrane was blocked for 30 min with blocking buffer. In cases where the primary antibodies were derived from different species, the peroxidase of the secondary antibodies was inactivated with 1% NaN3 in PBS for 45 min at RT.
2.2.9.5. Immunoprecipitation Washing buffer: 0.1% LM 0.1% NP-40 1 mM PMSF 50 mM Tris-HCl pH 7.4 10 nM NaF 0.1% NP-40 0.16 M NaCl
Immunoprecipitation (IP) is the technique of precipitating a protein antigen out of solution using an antibody that specifically binds to that particular protein. The antibody is coupled to a solid substrate such as protein A or protein G agarose beads. In our experiments, 40 x 106 cells were left untreated or stimulated with OT1-streptamers or CD3/CD8 mAbs for 3 min. Cells were then lysed in 500 µl ice-cold lysis buffer for 20 min on ice. After centrifugation (16,000 g, 10 min, 4°C), the
post-nuclear supernatant was taken and 2 μl of anti-ZAP-70 antibody as well as 30 μl protein-A agarose beads were added. To reduce non-specific binding, 1/10 volume of 10mg/ml BSA was added. The samples were incubated on a rotating wheel for 2 hours at 4°C. After washing 5x with 1 ml ice cold NP-40 washing buffer, the beads were incubated with 2x reducing loading buffer at 99°C for 5 min.
2.2.10. Immunofluorescence
12 spot slide (precoated with poly-L-Lysine)
Fixation solution: 3.5% PFA in PBS
Permeabilisation solution: 0.3% Triton X-100 in PBS
Blocking solution: 5% horse serum in PBS
Mounting medium: 2.4 g mowiol-488
6 g glycerol dissolved in 12 ml 0.2 M Tris (pH 8.5)
Cells were suspended in PBS/BSA (0.5%) at a concentration of 2 x 106/100 µl. 20 µl of the cell suspension was dropped per spot of a 12-spot slide and incubated at 4°C for 15 min to allow the cells to attach. Unattached cells were removed by washing in PBS. Cells were next fixed for 15 min followed by permeabilization for 10 min at RT. After washing for 3 times, the slide was blocked with 5% horse serum/PBS for 15 min at RT. The cells were then stained with the primary antibody for 60 min at RT. After 3 times washing, the cells were further incubated with the secondary antibody for 60 min in the dark at RT. Following three additional washing steps the samples were embedded in mounting media and the coverslips were fixed to the slide with nail polish. F-actin was detected by incubating with TRITC-phalloidin for 1 h at RT in the dark. The cells were imaged on a LEICA TCS SP2 laser-scanning confocal system and analyzed with the LEICAsoftware
3. Results
The affinity of the TCR for its ligand is one of the critical parameters regulating both thymocyte development and peripheral T cell activation (see Figure 8). In the thymus, intermediate affinity ligands induce survival and positive selection whereas high affinity stimuli direct thymocytes to apoptosis, a process known as negative selection. Thus, one receptor can induce two distinct cellular programs (differentiation or cell death). However, despite the progress in our understanding of the signaling pathways regulating T cell function, how the TCR can transmit signals leading to distinct cellular responses is so far not completely understood. Therefore, in this study, I utilized OT1-TCR tg mice as these mice possess a OT1-TCR of defined specificity. The OT1-OT1-TCR contains TCR-Vα2 and TCR-Vβ5 chains which recognize ovalbumin residues 257-264 (SIINFEKL) in the context of H2Kb. T cells were stimulated with two classical stimuli: (1) biotinylated CD3ε and CD8α mAbs that were cross-linked with streptavidin in solution and (2) the more physiological stimuli streptamers (Figure 13). OT1-streptamers are composed of biotinylated H-2Kb molecules loaded with the ovalbumin peptide and are cross-linked with Strep-Tactin. Both stimuli only bind the TCR and the costimulatory molecule CD8, however with different affinity and avidity. These cross-linked antibodies in solution have a much higher affinity (KD=2.4 x 10-9 M) than streptamers (KD=7 x 10-6 M) [93, 94] and, thus result in a much stronger stimulation of the cells.
Figure 13. Model of TCR triggering with either anti-CD3/CD8 mAbs or OT1-streptamers
CD8 positive T cells were either treated with soluble biotinylated CD3ε mAbs together with biotinylated CD8α mAbs, which were subsequently cross-linked with streptavidin or they were incubated with biotinylated H-2Kb molecules loaded with the high-affinity peptide SIINFEKL and cross-linked with Strep-Tactin (OT1-streptamers).
